Cycling of circadian rhythm proteins

描述(申请人提供)：长期目标是了解昼夜节律(~24小时)的分子基础。这些节律由大多数生物体内源性的时钟控制，并在许多不同的生理过程中表现出来。生物钟功能紊乱与睡眠障碍以及肿瘤生长等其他病理因素有关。内源性生物钟的分子性质在很大程度上是通过对果蝇黑腹果蝇的研究确定的。这些研究表明，时钟是由特定的基因(所谓的时钟基因)组成的，这些基因的蛋白质产物在一天中的特定时间调节自己的mRNAs的合成。由此产生的反馈回路驱动下游生理成分的循环，而下游生理成分反过来又驱动行为和生理的节奏。然而，维持这种以24小时为周期的反馈循环的机制尚不清楚。此外，在某些时钟mRNAs，有时甚至时钟蛋白的水平保持不变的条件下，经常保持节律，这表明翻译后调节的关键作用。果蝇的时钟与光的同步性也依赖于这种翻译后机制。我们假设，时钟蛋白的反馈活动必须循环才能维持时钟功能，并且这种活动的循环是由蛋白质稳定性、核表达和抑制转录的能力的时间控制驱动的。时钟蛋白的这些功能在很大程度上受到磷酸化的影响，但关键的调控步骤尚未确定。我们建议使用我们最近发现/产生的工具来剖析果蝇主要循环蛋白Period(PER)和TimeNever(TIM)的翻译后调节。我们还将研究时钟对光的反应，这是控制蛋白质稳定性的基础。具体目的是：(1)确定一个新的TIM等位基因行为表型的机制。我们发现了一个新的TIM突变，它影响PER和TIM的稳定性和核定位，以及PER的磷酸化。这为我们提供了一个强大的工具来研究核表达的潜在机制，并确定亚细胞定位对磷酸化和稳定性的影响；(2)确定新的磷酸化位点在PER和TIM上的功能意义。通过质谱分析，我们在PER和TIM上发现了新的磷酸化位点。我们将研究这些位点在上述过程中的作用；(3)确定TIM对光的反应所需的激酶。通过小分子抑制物筛选，我们已经确定了TIM在光下降解所需的各种类型的激酶。我们将确定涉及的特定激酶。

DESCRIPTION (provided by applicant): The long-term goals are to understand the molecular basis of circadian (~24 hour) rhythms. These rhythms are controlled by clocks endogenous to most organisms and are manifest in many different physiological processes. Disrupted functioning of clocks has been associated with sleep disorders as well as other pathologies such as tumor growth. The molecular nature of the endogenous circadian clock was determined largely through studies done in the fruit fly, Drosophila melanogaster. These studies showed that the clock is composed of specific genes (so-called "clock genes") whose protein products regulate the synthesis of their own mRNAs at a specific time of day. The feedback loop thus generated drives the cycling of downstream physiological components which, in turn, drive rhythms of behavior and physiology. However, the mechanisms that maintain such feedback loops with a 24 hour periodicity are not understood. In addition, rhythms are often maintained under conditions where levels of some clock mRNAs, and sometimes even clock proteins, are held constant, indicating the critical role of post-translational regulation. Synchrony of the Drosophila clock to light also relies upon such post-translational mechanisms. We hypothesize that it is the feedback activity of clock proteins that must cycle in order to maintain clock function, and that the cycling of this activity is driven by temporal control of protein stability, nuclear expression and ability to repress transcription. These features of clock proteins are affected, to a large extent, by phosphorylation, but key regulatory steps have not been identified. We propose to use tools we have recently discovered/generated to dissect the post-translational regulation of the major cycling Drosophila proteins, period (PER) and timeless (TIM). We will also investigate the clock's response to light, which has its basis in the control of protein stability. Specific aims are to: (1) Determine the mechanisms underlying the behavioral phenotype of a novel tim allele We have identified a novel mutation of tim which affects the stability and nuclear localization of PER and TIM and the phosphorylation of PER. This provides us with a powerful tool to address the mechanisms underlying nuclear expression, and to determine the effect of subcellular localization on phosphorylation and stability; (2) Identify the functional significance of novel phosphorylation sites on PER and TIM. We have identified novel phosphorylation sites on PER and TIM through mass spectrometry analysis. We will investigate the role of these sites in the processes listed above; (3) Identify kinases required for the TIM response to light. Through a small molecule inhibitor screen, we have identified classes of kinase required for TIM degradation by light. We will identify the specific kinases involved.